Project description:Increasing demand for the production of renewable fuels has recently generated a particular interest in microbial production of butanol. Anaerobic bacteria, such as Clostridium spp., can naturally convert carbohydrates into a variety of primary products, including alcohols like butanol. The genetics of microorganisms like Clostridium acetobutylicum have been well studied and their solvent-producing metabolic pathways characterized. In contrast, less is known about the genetics of Clostridium spp. capable of converting syngas or its individual components into solvents. In this study, the type of strain of a new solventogenic Clostridium species, C. carboxidivorans, was genetically characterized by genome sequencing. C. carboxidivorans strain P7(T) possessed a complete Wood-Ljungdahl pathway gene cluster, involving CO and CO(2) fixation and conversion to acetyl-CoA. Moreover, with the exception of an acetone production pathway, all the genetic determinants of canonical ABE metabolic pathways for acetate, butyrate, ethanol and butanol production were present in the P7(T) chromosome. The functionality of these pathways was also confirmed by growth of P7(T) on CO and production of CO(2) as well as volatile fatty acids (acetate and butyrate) and solvents (ethanol and butanol). P7(T) was also found to harbour a 19 Kbp plasmid, which did not include essential or butanol production related genes. This study has generated in depth knowledge of the P7(T) genome, which will be helpful in developing metabolic engineering strategies to improve C. carboxidivorans's natural capacity to produce potential biofuels from syngas.
Project description:Clostridium carboxidivorans P7 (DSM 15243) is a bacterium that converts syngas (a mixture of CO, H2, and CO2) into hexanol. An optimized and scaled-up industrial process could therefore provide a renewable source of fuels and chemicals while consuming industry waste gases. However, the genetic engineering of this bacterium is hindered by its multiple restriction-modification (RM) systems: the genome of C. carboxidivorans encodes at least ten restriction enzymes and eight methyltransferases (MTases). To gain insight into the complex RM systems of C. carboxidivorans, we analyzed genomic methylation patterns using single-molecule real-time (SMRT) sequencing and bisulfite sequencing. We identified six methylated sequence motifs. To match the methylation sites to the predicted MTases of C. carboxidivorans, we expressed them individually in Escherichia coli for functional characterization. Recognition motifs were identified for all three Type I MTases (CAYNNNNNCTGC/GCAGNNNNNRTG, CCANNNNNNNNTCG/CGANNNNNNNNTGG and GCANNNNNNNTNNCG/CGNNANNNNNNNTGC), two Type II MTases (GATAAT and CRAAAAR), and a single Type III MTase (GAAAT). However, no methylated recognition motif was found for one of the three Type II enzymes. One recognition motif that was methylated in C. carboxidivorans but not in E. coli (AGAAGC) was matched to the remaining Type III MTase through a process of elimination. Understanding these enzymes and the corresponding recognition sites will facilitate the development of genetic tools for C. carboxidivorans that can accelerate the industrial exploitation of this strain.